Comparison of a Real-time PCR for Detection of the tcdC Gene With Four Toxin Immunoassays and Culture in the Diagnosis of Clostridium difficle Infection

Division of Clinical Microbiology, Mayo Clinic, Rochester, Minnesota

^* To whom correspondence should be addressed. Email: rosenblatt.jon{at}mayo.edu.

	Abstract

We have developed a rapid real-time PCR method using fluorescence resonance energy transfer (FRET) probes and the LightCycler (Roche Diagnostics) which will detect from stool the presence of the tcdC gene of Clostridium difficile. Our PCR method also will identify the presence of base pair deletions, one of which (18 bp) has been associated with the "epidemic" toxin hyperproducing strains. We compared this PCR with three C. difficile toxin detecting enzyme immunoassays (EIAs), an EIA for detection of glutamate dehydrogenase (GDH), and with culture of C. difficile. A total of 200 stool specimens were studied by the comparative methods. C. difficile was isolated from 49 specimens by culture and 44 of these were confirmed as containing one of the genes associated with toxin production ("toxigenic culture"). Using toxigenic culture as the "gold standard", the sensitivities, specificities, positive and negative predictive values, respectively, of the assays were: Premier^TM Toxins A & B 48%, 98%, 88, 87%; ImmunoCard® Toxins A & B 48%, 99%, 91%, 87%; Xpect® C. difficile Toxin A/B 48%, 84%, 46%, 85%; Triage C. difficile Panel (for toxin A) 32%, 100%, 100%. 84%; LightCycler PCR 86%, 97%, 90%, 96%. Thus, in comparison to toxigenic culture, the sensitivities of the toxin immunoassays were unacceptably low while the LightCycler real-time PCR assay for detection of the tcdC gene of C. difficile is sensitive, and specific.